Meiotic double-strand break repair DNA synthesis tracts in Arabidopsis thaliana

We report here the successful labelling of meiotic prophase I DNA synthesis in the flowering plant, Arabidopsis thaliana. Incorporation of the thymidine analogue, EdU, enables visualisation of the footprints of recombinational repair of programmed meiotic DNA double-strand breaks (DSB), with ~400 discrete, SPO11-dependent, EdU-labelled chromosomal foci clearly visible at pachytene and later stages of meiosis. This number equates well with previous estimations of 200–300 DNA double-strand breaks per meiosis in Arabidopsis, confirming the power of this approach to detect the repair of most or all SPO11-dependent meiotic DSB repair events. The chromosomal distribution of these DNA-synthesis foci accords with that of early recombination markers and MLH1, which marks Class I crossover sites. Approximately 10 inter-homologue cross-overs (CO) have been shown to occur in each Arabidopsis male meiosis and, athough very probably under-estimated, an equivalent number of inter-homologue gene conversions (GC) have been described. Thus, at least 90% of meiotic recombination events, and very probably more, have not previously been accessible for analysis. Visual examination of the patterns of the foci on the synapsed pachytene chromosomes corresponds well with expectations from the different mechanisms of meiotic recombination and notably, no evidence for long Break-Induced Replication DNA synthesis tracts was found. Labelling of meiotic prophase I, SPO11-dependent DNA synthesis holds great promise for further understanding of the molecular mechanisms of meiotic recombination, at the heart of reproduction and evolution of eukaryotes.


Introduction
Meiosis is the specialised cell division that halves chromosome numbers during the production of gametes, essential for the maintenance of stable ploidy across generations in sexually reproducing organisms.In contrast to mitosis, in which duplication of the genome is followed by a single cellular division, meiosis involves one round of DNA replication followed by two rounds of division.Separating sister chromatids to opposite poles, the second meiotic division is, in broad terms, analogous to mitosis.Balanced chromosomal segregation during the first division however requires that homologous chromosomes recognise each other, pair and orient themselves on the spindle so that the two members of each pair move to opposite poles during anaphase.This is ensured by the programmed induction of homologous recombination through the induction and repair of DNA double-strand breaks (DSB) during the first meiotic prophase (reviews by [1][2][3]).
This programmed, highly regulated process of DNA damage and repair is essential for proper meiotic chromosome alignment and segregation and thus fertility in eukaryotes, with some known exceptions, notably Drosophila and C. elegans (see review by [4]).Following the programmed induction of DSB by the SPO11 complex, the cleaved DNA ends are resected to produce 3'-ssDNA overhangs, onto which the recombinases DMC1 and RAD51 are loaded to form presynaptic nucleoprotein filaments.These presynaptic nucleofilaments are the active molecular species in the search for, and invasion of an intact homologous DNA template, the first steps of DNA repair via homologous recombination.DNA synthesis then extends the invading 3' end across the template and the joint-molecule intermediate structures are further processed to yield the two main products of HR-mediated DSB repair: repair associated (gene conversion + crossover, CO), or not (gene conversion, GC), with reciprocal exchange of flanking sequences of the donor and recipient chromatids.For clarity, we note that only if the recombination involved non-sister chromatids, would the former be visible cytologically in meiosis as chiasmata and genetically as gene conversions ± crossovers (GC ± CO).In the same sense, the gene conversions would only be detectable if they include DNA sequence polymorphisms.
DNA synthesis is thus inherent to DSB repair via homologous recombination.In the 1960s, studies based on incorporation of H3-thymidine and C14-thymidine into newly synthesised DNA gave evidence for meiotic prophase I DNA synthesis in the plants Lilium longiflorum and Trillium erectum, the amphibian Triturum viridescens, mice and humans [5][6][7][8][9].These early studies have since been extended to show association of DNA synthesis with pachytene recombination nodules in Drosophila female meiosis and with the synaptonemal complex in mouse spermatocytes [10,11].
Labelling with 5-bromo-2 0 -deoxyuridine (BrdU) in budding yeast has been used to confirm the presence of SPO11-dependent meiotic prophase DNA synthesis [12].The authors characterized both single-molecule BrdU tracts at a recombination hotspot and the timing of their appearance in relation to other meiotic features.Observation of SPO11-dependent, BrdU labelling pattern has also been described in Tetrahymena [13] but, to our knowledge, no further data has been published in other organisms.In Arabidopsis, incorporation of BrdU or 5-ethynyl-2'-deoxyuridine (EdU) during pre-meiotic S-phase has been used to establish the meiotic timeline of meiosis in Arabidopsis pollen mother cells (PMC) [14][15][16].There are thus far no reports of labelling non-S-Phase DNA synthesis during Arabidopsis meiosis.
The induction of synchronous meiosis in populations of cells has been key to major advances in understanding of the molecular processes that make up the meiotic division [17][18][19][20][21].However, such direct approaches have not been available in other organisms and this has resulted in significant gaps in understanding.A clear example of this is seen in the flowering plant, A. thaliana.Estimations of numbers of SPO11-induced DSB in Arabidopsis pollen mother cells (PMC, male meiosis) are ~200-300 per meiosis.Of these, ~10 yield inter-homologue CO and another 10 have been shown to give inter-homologue gene conversions (GC) [22,23].Thus, the repair outcome of at least 90% of meiotic DSB in Arabidopsis is not known.The situation is strikingly different in budding yeast with 66 NCO plus 90.5 CO estimated per meiosis, the sum of which is very close to the estimated 140-170 meiotic DSB.Similarly in mouse, 273 NCO plus 27 CO sum close to the estimated 200-400 meiotic DSB [24][25][26][27].
Detection of recombination-associated DNA synthesis tracts offers an attractive path to understand the mechanisms underlying these "invisible" meiotic recombination events in Arabidopsis and potentially in other organisms.We present here the successful labelling of SPO11-dependent, meiotic prophase I DNA synthesis by EdU incorporation in Arabidopsis PMC.

EdU labelling of DSB repair-associated DNA synthesis
In Arabidopsis, while each inflorescence contains flower buds bearing PMC at different premeiotic and meiotic stages, the PMC of the anthers of a given flower bud are nearly synchronous [28,29].Along with the sequence of cytologically distinguishable meiotic stages in the buds of a given inflorescence, in practice it is relatively straightforward to classify PMC subpopulations within the meiotic timeline.
Timelines of Arabidopsis male meiosis concord that pre-meiotic G2 phase lasts 7-10 hours and that pollen mother cells advance from the beginning of leptotene to the end of pachytene in 19 to 21 hours [14][15][16]30] (summarized in Fig 1).Subsequent stages of meiosis are considerably faster, with post-pachytene meiosis estimated to take from 2.7 to 9 hours.Given that meiotic DSB formation begins at the onset of prophase I (early leptotene) and continues for a few hours [31], any meiotic prophase I DSB-repair DNA synthesis should occur at least 7-10 hours after the end of pre-meiotic S-phase.We thus decided to incubate the inflorescences with the nucleoside analogue for a 24-hour pulse to entirely cover the prophase I DSB repairassociated DNA synthesis window.Given the relative synchrony of progression through meiosis of the PMC from a given flower bud, the easily identifiable appearance of S-phase labelling and the possibility to easily situate subpopulations of meiotic cells in the temporal meiotic sequence, it seemed reasonable to expect that we would be able to exclude S-phase labelled meioses, and thus focus our analyses on those labelled during Prophase I.
Notwithstanding the current absence of direct evidence of the timing of occurrence of meiotic recombination intermediates in Arabidopsis, DAPI staining and immunolocalisation of meiotic proteins implicated in early invasion intermediates (RAD51/DMC1), joint molecules (MSH4, early HEI10) and resolution (MLH1, late HEI10) give a clear picture of the progression of key events through meiotic prophase I.In pachytene, when synapsis is complete, mean numbers of MLH1 foci correspond to the number of Class I crossovers (~90% of total CO), measured by counting of chiasmata at metaphase I.In analogy to organisms for which recombination intermediates can be tracked directly, it is reasonable to assume that most recombination events are either resolved or close to resolution at pachytene in Arabidopsis.
As summarised in Fig 1, inflorescences were thus incubated with EdU for 24 hours to ensure coverage of the window of DSB repair-associated DNA synthesis through prophase I up to the end of pachytene.Pachytene cells observed at the end of the 24-hour pulse would have traversed prophase I up to that point in the presence of the analogue.As mentioned above, nuclei with pre-meiotic S-phase labelling were easily identified and excluded from the analysis.

Non-replicative DNA synthesis tracts are present from pachytene until the end of meiosis
Early prophase I meiocytes, including cells in G2/leptotene and zygotene, showed complete or extensive EdU labelling of their chromosomes (Fig 2).This replicative labelling confirms, as expected, that they had passed through pre-meiotic S-Phase during the EdU pulse [14][15][16]32,33].Furthermore, nuclei with partial replicative labelling of DAPI-dense, late replicating heterochromatic regions were observed, as expected for cells that were in mid/late S-phase at the beginning of the EdU pulse (second row of Fig 2) [33][34][35][36].
In striking contrast, pachytene nuclei showed faint, discrete foci of a considerably smaller size (Fig 2 Pachytene, see also Figs 4A, S1 and S4).These foci are very faint through the microscope eyepiece, but are clearly visible when the images are processed.This difference in labelling intensity between S-phase and prophase EdU incorporation is not unexpected, considering that the latter would correspond to DNA synthesis tracts of tens to a few thousands of base pairs, while replicative DNA synthesis would range from megabase-long latereplicating regions to the whole genome (4C � 520 Mb in these prophase I nuclei) [37].We note also the presence of labelled mitochondria in these images, confirming their replication during pre-meiotic G2 and/or meiotic prophase I cells.These mitochondria and their characteristic changes in distribution, a standard feature of Arabidopsis meiotic spreads (for example, see [38,39]), are clearly visible from pachytene on.They are also present at earlier stages, but EdU labelling strategy and timeline of Arabidopsis male meiosis.Pollen mother cell nuclei were fixed and isolated at the end of a 24-hour pulse of EdU incorporation.Cells which traversed all or part of the pre-meiotic S-phase during the pulse will show replicative EdU incorporation and be at G2/leptotene/zygotene stages at the end of the pulse, with some overlap into early pachytene.Fully pachytene nuclei (mid-late pachytene) will not show replicative labeling, but will have traversed the leptotene and zygotene stages in the presence of EdU.Any EdU incorporation in these (or later) nuclei will be due to non-S-Phase DNA synthesis.https://doi.org/10.1371/journal.pgen.1011197.g001The EdU foci observed in pachytene cells appear well-distributed across the chromosomes, but are less present in DAPI-dense, heterochromatic regions (Fig 2 Pachytene).Furthermore, given the tighter-packed DNA in these regions, the observed lesser density of EdU foci is presumably an underestimation.This pattern of foci concords with genetic crossover mapping, studies of meiotic DSB (SPO11-oligo sequencing) and immunofluorescence studies of the distributions of early recombination proteins, all of which show meiotic recombination to follow a relatively even distribution across the Arabidopsis genome, except for (heterochromatic) centromeric regions which have significantly lower densities of SPO11-oligos and crossovers [37,40,41].
As summarised in Fig 1, based on published timelines [14][15][16]30] and our measurements, a meiocyte observed at the telophase II/tetrad stage would have been at leptotene/zygotene at the beginning of the 24-hour EdU pulse.We had thus good reason to expect that prophase I EdU foci would be observable in all meiotic stages up to the end of meiosis.This was indeed the case, with EdU foci visible from pachytene to the end of meiosis (telophase II/tetrad Fig 2).A clearly visible reduction in numbers of EdU foci and an increase in their size is seen as meiosis progresses.While we cannot formally exclude that these result from extension and fusion of the DNA synthesis tracts visible at Pachytene, we believe it is rather a visual artefact resulting from the progressive chromosomal condensation/compaction in meiosis.Meiotic chromosomes condense progressively as they traverse prophase I to Metaphase I (Fig 2 ), evolving from a mass of hardly-distinguishable thin fibres at early leptotene to thick synapsed fibres in pachytene and to individualised dense bivalents with a more polygonal shape at metaphase I.This condensation produces significant shortening of the chromosomes and results in changes in the visual character of the EdU signals.While most EdU foci at pachytene appear as discrete spots, they merge into larger patches at diplotene and this continues through diakinesis, up to the greatest condensation at metaphase I (Fig 2, diplotene-metaphase I).Furthermore, heterochromatic, centromeric regions become more easily identifiable when bivalents individualize at diakinesis and the relatively low density of EdU signals in these regions becomes clearly visible.This is even more pronounced at metaphase I, with clear visual differentiation between the centromeric and pericentromeric regions of the two homologous chromosomes of each bivalent.Finally, the EdU signals are observed throughout the second meiotic division, merging into larger patches with evolving chromosome condensation and shape in a way similar to that seen during the first meiotic division (Fig 2 , prophase II-tetrad).

Prophase I DNA synthesis is SPO11-dependent
Meiotic recombination is initiated by the induction of DNA DSB by the SPO11 complex at the beginning of meiotic prophase I. To verify that the discrete non-replicative DNA synthesis tracts observed at pachytene and subsequent stages originate from SPO11-dependent meiotic DSB repair, we carried out this analysis in parallel in spo11-1 mutant plants [42] (Fig 3).Pachytene images were analysed with the IMARIS software package (Bitplane AG, Oxford Instruments, UK) Spots function.EdU foci were selected based on diameter and mean intensity in the EdU channel and filtered to retain only those colocalising with chromosome fibres (mean intensity cutoff in the DAPI channel).A high-cut filter of mean intensity in the EdU channel was also applied to filter out the much brighter organellar foci.
WT pachytenes imaged with the Zeiss LSM 800/Airyscan confocal microscope show 412.1 ± 9.38 EdU foci (n = 35) per nucleus (mean ± SEM; n = 45 nuclei; Figs 2 and S1), with counts ranging from 323 to 510 (plus one outlier with 263).To confirm that we were not missing foci, ten image stacks were acquired via 3D-SIM for comparison.WT cells imaged with 3D-SIM had a mean 400.6 ± 14.74 EdU foci (n = 10) while those imaged with the confocal microscope had a mean of 412.1 ± 9.38 EdU foci (n = 35).No significant difference was thus observed between the counts from the two imaging systems (unpaired t-test; p = 0.55).Pooling the counts from both microscopes gives 409.5 ± 7.96 EdU foci per nucleus (mean ± SEM; n = 45 nuclei); Fig 4).
In spo11-1 plants, 39.2 ± 2.83 EdU foci per cell (mean ± SEM; n = 35 cells) were detected in zygotene/pachytene nuclei (Figs 3, 4 and S2).Thus, significantly lower numbers of EdU foci were detected in spo11-1 than in wild-type (WT) meioses.Similarly at subsequent meiotic stages, some EdU foci are detected in spo11-1 meioses, but in much lower numbers than the WT (Figs Fig 3, S2 and S3, Diakinesis to Telophase II).This ~10-fold reduction in mean numbers of foci with respect to the WT is highly significant (unpaired t-test; p < 0.0001) and confirms the SPO11-dependence of the great majority of the observed pachytene EdU foci.That the expected S-phase EdU labelling was observed in early prophase I spo11-1 PMC nuclei (Figs 3 and S3, early PI, mid PI, second row) and organellar EdU labelling was comparable in spo11-1 and WT meioses (Figs 2, 3 S2 and S3), confirms that the EdU labelling pulse worked as expected in both WT and spo11-1 plants.To facilitate visual comparisons between spo11-1 and WT at the different meiotic stages, the DAPI+EdU images from Figs 2 and 3 are presented together in S3 Fig.
In interpreting these numbers it is important to note that although Arabidopsis spo11 meiosis does complete and produce (mostly inviable) pollen, chromosome pairing and synapsis are very severely reduced in spo11 meiosis and the zygotene/pachytene stages are only identifiable by their characteristic chromosome condensation and their temporal positioning [42].Thus, spo11-1 PMC at leptotene can be differentiated from the zygotene-like state by the condensation of the chromosomes (thickness of the fibres) and the clustering of DAPI-dense regions.Also, spo11-1 Zygotene/Pachytene-like cells that do not show S-phase EdU labelling are in a more advanced state than those that do show it, as they would have completed S-phase at the beginning of the pulse (

Prophase I DNA synthesis patterns mirror meiotic recombination features
Meiotic recombination models predict different patterns of DNA synthesis tracts depending on how recombination intermediates are formed and resolved.Non-crossover events generated by either synthesis-dependent strand annealing (SDSA) or dHJ dissolution recombination, both expected to produce DNA synthesis tracts in only one of the two implicated chromatids and thus in only one of the homologous chromosomes in interhomologue recombination.On the other hand, dHJ resolution resulting in either crossover or non-crossover products would generate DNA synthesis tracts in both participating chromatids-and both chromosomes in interhomologue events.
The  Further examples of these patterns can be found in the pachytene images of S4 Fig.
Finally, we note the absence of the long labelling tracts expected from recombination through the break-induced replication pathway (BIR) [43], implying that such events are either rare or "short", or both, in Arabidopsis meiosis.

Prophase I DNA synthesis co-localize with class I crossovers
As the SPO11-dependent, meiotic prophase I DNA synthesis foci result from recombinational repair of meiotic DSB, genetic crossovers should co-localize with a subset of these EdU foci.Class I CO are detectable cytologically in Arabidopsis by the immunolocalization of proteins such as MLH1.
Combining labelling of EdU tracts with immunolocalization of MLH1 (Fig 6) gave 7.86 ± 0.24 (mean ± SEM; n = 29) MLH1 foci per WT pachytene, as expected [44].95.6% (n = 228) of all MLH1 foci co-localized with an EdU focus and the remaining 4.4% were adjacent to one.Although this conclusion must be tempered due to the high number of EdU foci

Identification of meiotic DNA repair-associated DNA synthesis tracts
We present here the labelling and characterisation of SPO11-dependent, meiotic prophase I DNA synthesis tracts through incorporation of the thymidine analogue EdU in pollen mother cells of Arabidopsis plants.By labelling DNA synthesis associated with the repair of meiotic DSB, this approach holds great promise for the future understanding of recombination patterns and mechanisms, notably for studies of NCO and sister-chromatid exchange (SCE) recombination.For practical reasons, molecular and genetic studies of meiotic recombination have essentially been focussed on events implicating the transfer of DNA sequence polymorphisms between the two recombining DNA molecules.The necessary presence of such polymorphism has, for instance, complicated significantly the analysis of the roles of meiotic SCE [45][46][47][48][49][50][51][52] and while EdU labelling has recently been applied to analysis of meiotic sister-chromatid CO in C. elegans [53][54][55], this approach does not appear adapted to the study of NCO.
Detection of NCO events in Arabidopsis has been elusive.Mapping inter-homologue gene conversions (GC) genome-wide by sequencing Arabidopsis tetrads has estimated their numbers to be of the same order as CO, ~10 events per meiosis, with gene conversion tracts ranging between a few tens to a few hundreds of bp [22,23,56,57].Notwithstanding this (surprisingly) low number of inter-homologue GC, numbers of CO agree with chiasmata counting and immunolocalisation of CO markers.Thus, given the 200-300 DSB per meiosis in these cells, the repair of at least 90% (and very possibly >98% [39] of meiotic DSB in Arabidopsis is not accessible to analysis with current methods.In this work, we present the development of meiotic prophase DNA synthesis labelling as a means to fully characterise meiotic DSB repair in Arabidopsis meiocytes. Initial tests showed that incubating inflorescence stems for 24 hours in EdU solution resulted in two clear sub-populations of meiocytes, distinguishable by their meiotic stage and pattern of EdU labelling.The first consists of late G2 + early prophase I cells (leptotene, zygotene) whose chromosomes show very strong EdU signals, either genome-wide or in large patches.These patterns are consistent with those expected from pre-meiotic S-phase replicative EdU incorporation: I.This extensive EdU labelling is observed in meioses of both wild-type and spo11 mutant plants.
II.The extensive and intense fluorescent signals are similar to those observed in previous reports of EdU/BrdU incorporation during pre-meiotic replication [14][15][16]32].
III. Extrapolating from published meiotic timelines and our studies, PMC in pre-meiotic Sphase at the beginning of the pulse are expected to advance at most to leptotene-zygotene during the 24-hour EdU pulse (see Fig 1).
IV.The signal in partially labelled early prophase I cells corresponds principally to late-replicating, DAPI-dense, heterochromatic regions, in accordance with expectations for cells in late S-phase at the beginning of the EdU pulse.
The second population of EdU-labelled meiocytes includes cells at meiotic stages from pachytene to the end of meiosis (tetrads), whose chromosomes show much fainter, discrete EdU labelling.Clearly resolving into individual foci at the stages in which the chromosomes are less condensed, these foci group into "patches" as chromosome condensation proceeds post-prophase I.These foci have a number of characteristics which permit their identification with confidence as meiotic DSB repair-associated DNA synthesis tracts: I.These discrete EdU-label foci are SPO11-dependent, analogously to earlier observations in yeast [12], confirming with high confidence that they result from EdU incorporation at DNA synthesis tracts associated with meiotic DSB repair.Some EdU foci are observed in spo11-1 meioses, although at considerably lower numbers than in the wild type (39 versus 410 per meiosis respectively).While some of these may come from mistaken assignation of organellar DNA foci, we note that some crossing-over is detected in Arabidopsis spo11 plants, RAD51 foci are detected in Arabidopsis spo11 meiosis and DMC1 foci in rice meioses [42,[58][59][60].Furthermore, given that both meiotic Arabidopsis SPO11 proteins are needed for normal meiotic DSB induction, some leakage may occur in plants lacking one or the two SPO11 proteins [59,[61][62][63][64].Alternatively, they may correspond to SPO11-independent breakage of mechanical, chemical or enzymatic origin.If so, that they do produce DNA neo-synthesis tracts argues for their repair by HR rather than End Joining.
II.The SPO11-dependent foci are seen at later meiotic stages than those with replicative labelling and these nuclei had thus completed S-phase prior to the beginning of the pulse.Given the known timing of meiosis in these cells, meiotic DSB repair DNA synthesis will have occurred during the EdU pulse in these cells [12,16,17,44,65].
III.The discrete EdU foci observed at Pachytene and later stages are both much less intense and smaller in size than the generalised labelling in Leptotene-Zygotene nuclei.This is expected given the predicted size of DNA synthesis tracts associated with recombination events (few bases to some kilobases of DNA), with respect to replicative labelling (megabases to the full genome).
IV.The numbers of EdU foci measured at pachytene concord with published estimations of numbers of meiotic DSB and HR intermediates in Arabidopsis.A mean of 409.5 ± 7.96 (mean ± SEM; n = 45 meioses) DNA repair-associated DNA synthesis foci were observed in WT pachytenes.This number is a little higher than estimations of meiotic DSB numbers through immunolocalisation of early recombination proteins in Arabidopsis (RAD51 in most cases; 90 to 250 foci per meiosis) and compare well with a recent report of 239 ± 30 (mean ± SD) prophase I SPO11-1 foci [61].Given the limitations of immunodetection and that the recruitment/release of these proteins at DSB/recombination sites is dynamic, these numbers presumably under-estimate the true values.Notably, a recent report using STED super-resolution microscopy found more than 1000 RAD51 foci per PMC meiosis [39].Thus, the mean of 409.5 DNA repair-associated synthesis tracts per meiosis reported here is probably closer to the true value than that of protein-immunodetection approaches.It is, of course, necessary to take into account the fact that resolution of dHJ recombination intermediates is expected to result in patches of neo-synthesised DNA on both implicated chromatids, resulting in two foci from a single recombination event.Such events are, however, estimated to represent ~5% or less of meiotic DSB repair events in Arabidopsis and so are not expected to impact significantly the estimations of total events.On the other hand, it is also possible that very short or poorly labelled tracts may not give detectable foci under the microscope, leading to underestimation of true numbers of DNA synthesis tracts.
V. DAPI-dense heterochromatic, centromeric regions have a visibly lower density of SPO11dependent EdU foci in pachytene/diplotene and beyond (particularly noticeable at diakinesis/MI).This concords with SPO11-oligo mapping of meiotic DSB patterns in Arabidopsis PMC, which shows that centromeric regions have a much lower density of DSB than the rest of the chromosome [37,41].
VI. Combining labelling of EdU tracts with immunolocalization of MLH1 confirms that, as expected, Class I CO are physically associated with the prophase I DSB repair DNA synthesis tracts (Fig 6).We note, however, that the high number of EdU tracts spread across the pachytene SC fibres in these images means that some care should be taken with this conclusion.

Meiotic DSB repair-associated DNA synthesis tracts mirror meiotic recombination features
As presented in Fig 7, different mechanisms of recombinational repair of meiotic DSB will result in different patterns of DNA synthesis tracts associated with an HR event.The most obvious difference is that DNA synthesis is expected to be found on only the recipient While this outcome can be altered by migration of one or both Holliday Junctions, in both cases, this can lead to the presence of neo-synthesised DNA on both the donor and recipient chromatids.These events would thus be expected to result in paired DNA synthesis tracts on the two participating chromatids: either on one chromosome (Class (2)-one lateral axis) for sister chromatid events, or on both homologues (Class (3)-both lateral axes).Class (2) are thus "pairs" of foci on one of the two homologues (one SC lateral axis), which could result from recombination between sister chromatids through the dHJ resolution pathway.Class (3) are "pairs" of foci on the two homologues (both lateral axes), which could result from recombination between homologues through the dHJ resolution pathway.
We find no evidence for long DNA synthesis tracts indicative of break-induced replication (BIR) recombination [43] in our images.Thus, either BIR replication tracts are not distinguishably longer than other DSB repair-DNA synthesis, or the BIR mechanism is a minor (or non-) contributor to DSB repair in Arabidopsis meiosis.In this context, we note that an earlier effort to detect mitotic BIR repair in bleomycin-treated Vicia faba root tips described asymmetric interstitial G2-phase EdU labelling of interstitial tandem-repeat regions, although no labelling tracts extending out to the end of the chromatid were observed [66].We also note that the equivalence of numbers of DSB and DNA neo-synthesis tracts argues for repair by HR rather than End Joining, which would not be expected to produce detectable DNA synthesis.
Inference of the recombination pathways underlying the 3 classes of foci, of course, remains speculative in the absence of molecular analyses of the EdU tracts themselves.Unequivocally distinguishing "true" paired foci with a common mechanistic origin, from simple proximity resulting from the high number of EdU foci present on the relatively limited surface on the pachytene fibres would be very difficult.FISH on extended DNA fibres was used in an earlier study in yeast to determine the lengths and positioning of BrdU-substituted DNA synthesis tracts with respect to DSB hotspots [12].They observed two main classes of tracts: tracts that extended from the DSB towards only one side of the DSB and tracts that extended on both sides.These two classes correspond to the tracts expected in single DNA molecules that took part in CO events in the first case and NCO events via SDSA in the second, which were further validated studying their dynamics in recombination mutants.We have previously carried out DNA combing plus fibre FISH in Arabidopsis [67], but the lack of strong meiotic DSB hotspots with a precisely localised DSB site complicates the possible use of this approach in Arabidopsis meiosis.Rather, we are actively developing an approach based on the direct detection of EdU-substituted DNA tracts by Nanopore sequencing.It is hoped that this approach will permit unambiguous characterisation of the principal meiotic recombination products and underlying mechanisms in Arabidopsis, as well as of more complex events.

Plant material and growing conditions
Standard conditions were used for growth of wild-type A. thaliana Col-0 and spo11-1-2 [42] plants.Seeds were sown on soil substrate (Klasmann-Deilmann GmbH TS3 FIN 416GF, Geeste, Germany), stratified for 2-4 days at 4˚C and grown in climate chambers at 23˚C and 60% relative humidity, under a daily cycle of 16 hours of light (110-140 μmol m -2 s -1 ) and 8 hours of darkness.

Meiotic chromosome preparation by spreading
Meiosis was analysed following the chromosome spreading method of Ross et al. [38], with minor modifications.Inflorescences were collected in 3:1 ethanol:glacial acetic acid fixative solution and left overnight at room temperature.The fixative was then substituted with fresh fixative solution and the tubes were transferred to 4˚C.This step was repeated the following 2-3 days.At this point, fixed inflorescences can be stored at 4˚C for further use or prepared for microscopy.
Several fixed inflorescences were selected, deposited in a glass dissection well with 3:1 fixative solution and dissected under a stereomicroscope into individual flower buds, discarding open flowers and buds with pollen (yellow anthers).The flower buds were then washed 3 x 2 minutes in citrate buffer and the buffer replaced with 500 μl of digestion enzyme mix (0.3% w/ v cellulase from Trichoderma sp.(Sigma-Aldrich), 0.3% w/v pectolyase from Aspergillus japonicus (Sigma) and 0.3% w/v cytohelicase from Helix pomatia (Duchefa Biochemie) in citrate buffer).The flower buds were incubated in the digestion mix for 2 hours at 37˚C and the digestion stopped by adding a greater volume of ice-cold citrate buffer.The well was placed under the stereomicroscope and a single flower bud with a small volume of liquid was transferred to a slide with a Pasteur pipette.The flower bud was macerated with a needle, a 10μl drop of icecold 60% glacial acetic acid was added and the slide incubated for one minute on a hot plate at 45˚C while gently moving the drop with a flat needle.After the incubation, an extra 10 μl of ice-cold 60% glacial acetic acid was added, followed by 100 μl of 3:1 fixative-first circling and flooding the drop and then mixing with the drop by vigorously pipetting the last microliters.The liquid was drained by tilting the slide, which was then further washed with another 100 μl of 3:1 fixative, drained and left to air dry at room temperature.
To visualise the chromosomes, the slides were stained in Vectashield antifade mounting medium with 1.5 μg/ml DAPI (# H-1200, Vector Laboratories, Burlingame, CA, USA), by putting a drop on a coverslip which was then placed on a slide and gently squashed by thumb pressure.Spreads were analysed with a Zeiss AxioImager Z1 microscope with a 100×/1.40Oil Plan-Apochromat objective and the Zeiss ZEN2Blue software (Carl Zeiss GmbH)in the channel for blue fluorescence (Zeiss #49 HE filter).

EdU labelling and cytological detection
Among the different thymidine analogues commonly used to label in vivo DNA synthesis, 5-Ethynyl-2'-deoxyuridine (EdU) has a number of advantages and has been preferred by the Arabidopsis community in recent studies labelling DNA synthesis [15,32,33,[68][69][70][71].EdU can be detected in intact double-strand DNA and this antibody-free technique simplifies the protocol and offers better reproducibility.
EdU (#Ab146186, Abcam, UK) working solutions were prepared at 10mM in 1×PBS buffer.Arabidopsis stems with apical inflorescences were submerged in water to avoid the introduction of air bubbles (submerging only the stem segment to be cut, without wetting the inflorescences) and cut obliquely 4-5cm below the inflorescence.Care was taken to make the cleanest cut possible to avoid crushing vascular tissues.Open flowers and big leaves (if present) were removed to avoid crowding the tube and minimise the risk of exhausting the solution due to excessive transpiration.The inflorescences were incubated in 800μl of 10mM EdU in a 2ml Eppendorf tube for 24 hours, under the same growing conditions as the plants.To avoid tight packing, a maximum of four or five inflorescence stems were incubated in one tube.After 24 hours the inflorescences were removed from the EdU solution and fixed in 3:1 ethanol:glacial acetic acid solution.
Chromosome spreads were prepared as previously described and visualised under the microscope to identify those slides with meiocytes at the desired meiotic stages.The slides were first incubated in 4T (4×SSC, 0.5% v/v Tween 20) in a Coplin jar until the coverslips detached, followed by 30' without coverslips in fresh 4T.The Click-iT reaction mix (Invitrogen Click-iT plus Alexa-fluor 647 kit, Thermo-Fisher Scientific) to label EdU with the fluorochrome was prepared following the manufacturer's instructions.Before labelling, the slides were washed for 5 minutes with 1×PBS.A 30μl drop of Click-iT reagent was placed on a 32x24mm piece of parafilm and the inverted slides were placed on top of the mix on the parafilm.The slides were incubated with the mix for 30 minutes at 37˚C in a dark moist chamber, washed for 5 minutes with 1×PBS in a Coplin jar in the dark and mounted in Vectashield + DAPI for microscopy.
Pictures were taken using a Zeiss LSM800 confocal microscope applying the Airyscan module and selecting the presets for each specific dye.EdU foci were quantified semi-automatically using the IMARIS software Spots tool (Bitplane AG, Oxford Instruments, UK), adding intensity filters for both channels to specifically selected visually detectable EdU foci that co-localised with the DAPI fluorescence of the chromosomes.To analyse the chromatin and EdU signal ultrastructure, we applied super-resolution spatial structured illumination microscopy (3D-SIM) using a 63×/1.40Oil Plan-Apochromat objective of an Elyra PS.1 microscope system and the ZENBlack software (Carl Zeiss GmbH) [72].Maximum intensity projections were calculated from 3D-SIM image stacks.Zoom-in sections were presented as single slices to indicate the subnuclear chromatin structures at the super-resolution level.

Detection of MLH1 and EdU
For the detection of MLH1 and neo-synthesised DNA tracts with incorporated EdU, the protocol for the immunolocalization of MLH1 [73] was adapted to include the Click-iT EdU labelling.Chromosome spreads were prepared as previously described and visualised under the microscope to identify those slides with meiocytes at the desired stages.The slides were incubated in a Coplin jar with 100% ethanol to detach the coverslip and washed with 1×PBST (0.1% Triton X-100 in 1×PBS.Another glass staining jar was filled with tri-sodium citrate buffer pH7 and heated to boiling point in a microwave.The slides were placed in the heated tri-sodium citrate solution for 45 seconds, after which they were returned to room temperature 1×PBST. The rabbit anti-MLH1 primary antibodies (kindly provided by Mathilde Grelon, INRAe Versailles [44]) were prepared at 1/200 in 1×PBST + 1% BSA.A 45μl drop was placed on a 32x24mm piece of parafilm and the inverted slide place on this.The slides were incubated for 2 days at 4˚C in a dark moist chamber, after which they were washed 3 x 5 minutes with 1×PBST.The secondary antibodies (goat anti-rabbit Alexa fluor 488) were prepared at 1/100 1×in PBST + 1% BSA, and 45μl were used per slide.Slides were incubated 30 minutes at 37˚C in a dark moist chamber, after which they were washed 2 x 5 minutes in 1×PBST, followed by one wash with 1×PBS.The Click-iT reaction was then performed as described above and the slide was mounted in Vectashield + DAPI mounting medium for microscopy.Counting of MLH1 foci and co-localization with EdU foci was carried out by visual inspection of images taken using the Zeiss LSM800 confocal microscope plus the Airyscan module.

Fig 1 .
Fig 1.EdU labelling strategy and timeline of Arabidopsis male meiosis.Pollen mother cell nuclei were fixed and isolated at the end of a 24-hour pulse of EdU incorporation.Cells which traversed all or part of the pre-meiotic S-phase during the pulse will show replicative EdU incorporation and be at G2/leptotene/zygotene stages at the end of the pulse, with some overlap into early pachytene.Fully pachytene nuclei (mid-late pachytene) will not show replicative labeling, but will have traversed the leptotene and zygotene stages in the presence of EdU.Any EdU incorporation in these (or later) nuclei will be due to non-S-Phase DNA synthesis.

Fig 2 .
Fig 2. DSB repair-associated DNA synthesis tracts are detected from pachytene up to the end of meiosis.Meiotic stages in A. thaliana wildtype.S-phase, replicative DNA labelling is seen at early G2/letotene and zygotene stages, while clear, discrete foci of EdU substituted DNA are visible in the chromosomes at later stages from pachytene onwards.Rectangles indicate the enlarged regions presented in the zooms.Non-chromosomal EdU foci correspond to labelling of mitochondrial genome replication.3μm scale bars are shown at the bottom left of each image (1μm for the zooms).Images were taken with the confocal microscope + Airyscan module.https://doi.org/10.1371/journal.pgen.1011197.g002

Fig 3 .
Fig 3. DSB repair-associated DNA synthesis is SPO11-dependent.Meiotic stages in A. thaliana spo11-1 mutants.As for WT meiosis (Fig 2), replicative S-phase labelling is seen at early stages, corresponding to G2/leptotene and zygotene.As expected, a striking reduction in numbers of EdU foci (compared to WT) is observed in spo11-1 mutants at stages from mid-prophase I onwards (corresponding to pachytene and later stages).Rectangles indicate the enlarged regions presented in the zooms.Images taken with the confocal microscope with Airyscan module.3μm scale bars are shown at the bottom left of each image (1μm for the zooms).https://doi.org/10.1371/journal.pgen.1011197.g003 Fig 3, mid-Prophase I vs early-mid Prophase I).Identification of later meiotic stages in spo11-1 meioses presents no difficulties (Fig 3).
expected patterns of resulting EdU foci are shown schematically in Fig 5A and examples of segments of the synaptonemal complex (SC) from SIM pachytene micrographs are shown in Fig 5B.The paired, homologous chromosomes are clearly distinguishable in these images, permitting a first analysis of different patterns of EdU foci.No obvious bias is visible in the disposition of Individual EdU foci in these images.Foci are seen at both the internal (facing the homologous chromosome) and external faces of the paired chromosomes in the SC fibre, as well as in the middle of the SC (Fig 5).Differences can however be seen when EdU foci are analysed with respect to adjacent foci, with three clear configurations of pairs of foci.Examples of which are circled on the images shown in Fig 5B:

Fig 4 .
Fig 4. Concordance of numbers of SPO11-dependent, meiotic DNA synthesis foci and estimated DSB numbers.a) Representative images of wild-type pachytene and spo11-1 pachytene-like meiotic stages.The merged DAPI (blue) plus EdU (orange) images are accompanied by computer-generated images of the scored EdU foci (white, right panels).EdU foci colocalising with chromosome fibres were retained for counting and mean intensity used to exclude the much-brighter organellar foci (see text).The images taken with the confocal microscope with Airyscan module.3μm scale bars are shown at the bottom left of each image.b) Numbers of EdU foci on the chromosomes quantified with Imaris software show clearly the SPO11-dependence of these DNA synthesis tracts and that their numbers correspond to expectations from DSB counting in PMC.The mean number of foci/nucleus and the number of nuclei counted are shown above the graph.Data used for the plot in (b) is presented in the S1 Data File.https://doi.org/10.1371/journal.pgen.1011197.g004

Fig 5 .
Fig 5. Ultrastructure of SPO11-dependent, meiotic DNA synthesis foci on the synaptonemal complex (SC) during pachytene.a) Expected configurations of DNA synthesis tracts resulting from different mechanisms of homologous recombinational repair of meiotic DSB: Synthesis-Dependent Strand Annealing (SDSA), double-Holliday Junction (dHJ), Gene Conversion ± Crossover (GC±CO) and Break-Induced Replication (BIR).b) Examples of segments of pachytene SC.Isolated individual foci (one SC lateral axis, white circle) and pairs of foci on one homologue (one SC lateral axis, green circle) or the two homologues (both SC lateral axes, yellow circle) are highlighted.The images represent a single slice of 3D-SIM image stacks.Scale bars are shown at the bottom left of each image.https://doi.org/10.1371/journal.pgen.1011197.g005

Fig 6 .
Fig 6.Class I crossovers (CO) co-localize with prophase I DNA synthesis tracts.a) Wild-type pachytene cell showing DAPI staining (blue), prophase I DNA synthesis tracts (EdU, orange) and Class 1 CO (MLH1, green).b) Examples of colocalising and adjacent MLH1 and EdU foci.The quantification (right) shows that 95.6% of MLH1 foci co-localize with EdU tracts, and the remainder are adjacent to one.Confocal images.Scale bars are 1μm in (a) and 0.1μm in (b).Data used for the plot in (b) is presented in the S1 Data File.https://doi.org/10.1371/journal.pgen.1011197.g006

Fig 7 .
Fig 7. Recombination mechanisms and DNA synthesis tracts.Following resection and invasion of the donor chromatid (a), DNA neosynthesis tracts resulting from repair of DSB are expected to be found only on the recipient chromatid in gene conversions (f) resulting from SDSA (b) or dHJ dissolution (e) pathways.A special case of this is the BIR pathway (c), which will result in a long synthesis tract, potentially extending all the way to the end of the recipient chromatid.DNA synthesis tracts are expected to be found on both the recipient and donor chromatids in gene conversions, associated with a CO (g) or not (h), resulting from resolution of the dHJ intermediate (d).https://doi.org/10.1371/journal.pgen.1011197.g007